Project Summary Transposable elements (TEs) play important roles in evolution of genomes, but their long-hypothesized role in development remains highly controversial. Indeed, despite possessing sophisticated epigenetic and post- transcriptional mechanisms of TE repression, the germline as well as various somatic and cancer cells express TEs at least once during their di?erentiation. However, whether TEs contribute to cell di?erentiation is shrouded in mystery. Numerous studies over the past decade demonstrated that overexpression of retrotransposon LINE-1, the only autonomously active TE in the human genome, is toxic to cultured human cells as well as to mouse germ cells with disrupted epigenetic repression of TEs. Furthermore, our recent studies of the conserved phenomenon of fetal oocyte aGrition have implicated excessive LINE-1 expression in providing the basis for the selective culling of mouse oocytes. Since epigenetic mechanisms are crucial for TE repression as well as normal development, these observations suggest that LINE-1 contributes to development by eliminating cells whose epigenetic remodeling during cell di?erentiation exceeds their capacity to restrain reactivated TEs. I Which aspects of LINE-1 activity trigger cytotoxicity? LINE-1-encoded ORF2p has two enzymatic activities, Endonuclease (EN) and Reverse Transcriptase (RT), that are required for retrotransposition. LINE-1- driven DNA damage clearly depends on EN activity, but the contribution of the RT activity to cytotoxicity is unknown. We have now identi?ed a robust model system for the in vivo analysis of LINE-1 RT impact on the host, namely, meiotic prophase (MP) fetal oocytes of mice that express LINE-1 following epigenetic reprogramming of their genomes. Importantly, LINE-1 expression levels vary between individual oocytes and correlate with oocyte viability, leading to the loss of 50% of oocytes in the early MP, before the activation of the DNA damage checkpoint. Furthermore, administration of an RT inhibitor AZT prevents oocyte lethality in the early MP implicating RNA:DNA hybrid intermediates of LINE-1 retrotransposition in triggering oocyte death in a DNA damage-independent manner. I The proposed research will identify and quantify L1 RNA:DNA hybrids in AZT treated and untreated fetal oocytes of wild type and L1 overexpressing mice, and establish a role for L1 RNAL:DNA hybrids in RNase H2 de?cient mice. Overall, the proposal will reveal a previously unknown mechanism of L1 RT- dependent death of fetal oocytes which could lead to deeper exploration of this phenomenon in somatic and cancer cells.